The present invention relates to the immobilization of nucleic acids. In another aspect, the invention relates to solid supports, such as oligonucleotide (xe2x80x9coligoxe2x80x9d) chips, incorporating such nucleic acids. In yet another aspect, the invention relates to photoreactive groups, to molecules derivatized with such groups, and to the attachment of such molecules to support surfaces by the activation of such groups.
The development of oligonucleotide (xe2x80x9coligoxe2x80x9d) probe arrays (more commonly known as xe2x80x9cDNA chipsxe2x80x9d and xe2x80x9cGene Chipxe2x80x9d (a registered trademark of Affymetrix, Inc.)) has made significant advances over the past few years, and is becoming the center of ever-increasing attention and heightened significance. See, for instance, Stipp, D., Fortune, p.56, Mar. 31, 1997. See also Borman, S., CandEN, p.42, Dec. 9, 1996, and Travis, J., Science News 151:144-145 (1997).
Typically, oligonucleotide probe arrays display specific oligonucleotide sequences at precise locations in an information rich format. In use, the hybridization pattern of a fluorescently labeled nucleic acid target is used to gain primary structure information for the target. This format can be applied to a broad range of nucleic acid sequence analysis problems including pathogen identification, forensic applications, monitoring mRNA expression and de novo sequencing. See, for instance, Lipshutz, R. J., et al., BioTechniques 19(3):442-447 (1995). Such arrays sometimes need to carry several tens of thousands, or even hundreds of thousands of individual probes. The chips also need to provide a broad range of sensitivities in order to detect sequences that may be expressed at levels anywhere from 1 to 10,000 copies per cell.
A variety of approaches have been developed for the fabrication and/or use of oligonucleotide probe arrays. See, for instance, Weaver, et al. (WO 92/10092) which describes a synthetic strategy for the creation of large scale chemical diversity on a solid-phase support. The system employs solid-phase chemistry, photolabile protecting groups and photolithography to achieve light-directed, spatially addressable, parallel chemical synthesis. Using the proper sequence of masks and chemical stepwise reactions, a defined set of oligos can be constructed, each in a predefined position on the surface of the array.
Using this technology, Affymetrix, Inc. (Santa Clara, Calif.), has developed large microarrays of oligonucleotides affixed to silicon wafers. In use, a researcher will extract mRNA from a cell or other biological source, convert it to cDNA and label the sample with a fluorescent probe. Sequences complimentary to the chip-bound probe will hybridize to the wafer and allow the researcher to determine their relative amounts by measuring the fluorescence of each spot. To date, for instance, researchers have been able to quantitatively measure the expression of more than 1000 human genes in this manner.
One drawback of the Affymetrix approach is the limitation of the oligo length that can be affixed to the surfaces. With present techniques, it is common that every addition step involved in the synthesis of oligos will result in some errors or truncated sequences. With oligo chips, however, it is not possible to perform conventional post-synthesis purification techniques (e.g., HPLC) in order to remove truncated sequences since the oligo sequences remain bound to the support.
Synteni (Palo Alto, Calif.) produces arrays of cDNA by applying polylysine to glass slides. Arrays of cDNAs are printed onto the coated slides followed by exposure to UV light, in order to crosslink the DNA with the polylysine. Unreacted polylysine is then blocked by reaction with succinic anhydride. These arrays, called xe2x80x9cGene Expression Microarraysxe2x80x9d (GEM(trademark)) are used by labeling cDNA prepared from a normal cell with a fluorescent dye, then labeling cDNA from an abnormal cell with a fluorescent dye of a different color. These two labeled cDNA probes are simultaneously applied to the microarray, where they competitively bind to the arrayed cDNA molecules. This two color coding technique is used to identify the differences in gene expression between two cell samples. (Heller, R. A., et al., Proc. Natl. Acad. Sci. USA, 94:2150-2155 (1997)).
Others have described a photo-crosslinking compound known as psoralen. Psoralen is a polycyclic compound having a planar configuration that intercalates within nucleic acid helices. When irradiated with UV light, the intercalated psoralen induces the formation of inter-strand linkages within the DNA molecule. Oligonucleotides derivatized at the 5xe2x80x2-terminus with psoralen have been used to crosslink double-stranded (Pieles, U. and U. Englisch, Nucleic Acid Res., 17(1):285-299, 1989), or triplex nucleic acids in solution (Takasugi, M., et al., Proc. Natl. Acad. Sci. USA, 88(13):5602-5606 (1991)). Psoralen derivatives have also been used to crosslink DNA-binding proteins to DNA (Sastry, S. S., et al., J. Biol. Chem., 272(6):3715-3723 (1997)).
In a different application, psoralen derivatives have been used to covalently bond functional groups to the surface of solid supports such as polystyrene. Those functional groups, in turn, are then used to thermochemically attach compounds to the support surface (Goodchild, J., Bioconjugate Chem., 1(3):165-187 (1990)). Currently, Nalge Nunc International uses this process to produce microplates that provide amine-functionalized surfaces for the thermochemical attachment of molecules such as nucleic acids. See, e.g., xe2x80x9cDNA Assay Developments: Surface Chemistry and Formats for Molecular Screening and Diagnosticsxe2x80x9d, B. Sullivan, et al., Jun. 4, 1997, Nalge Nunc International Corporation product literature.
On a separate subject, the assignee of the present invention has previously described a variety of applications for the use of photochemistry, and in particular, photoreactive groups, e.g., for attaching polymers and other molecules to support surfaces. See, for instance, U.S. Pat. Nos. 4,722,906, 4,979,959, 5,217,492, 5,512,329, 5,563,056, 5,637,460, and 5,714,360 and International Patent Application Nos. PCT/US96/08797 (Virus Inactivating Coatings), PCT/US96/07695 (Capillary Endothelialization), and PCT/US97/05344 (Chain Transfer Agents).
To the best of Applicant""s knowledge, however, the art does not teach, nor are there commercial products that involve, the activation of pendent photoreactive groups to attach nucleic acids to surfaces in a specific and controllable fashion. The attachment of a nucleic acid to a surface by irradiation would appear to be susceptible to radiation-induced damage, and would be inherently nonspecific. See, for instance, M. Pirrung, et al., J. Org. Chem., 63:241-246 (1998), which states that xe2x80x9cirradiation [during deprotection] with wavelengths less than 340 nm should be avoided . . . based on the potential photochemical damage to the DNA.xe2x80x9d
In spite of the developments to date, there remains a need for methods and reagents that improve the immobilization of nucleic acids onto a variety of support materials, e.g., in order to form oligonucleotide probe arrays.
The present invention provides a composition comprising a photoactivatable nucleic acid derivative, in the form of a nucleic acid having one or more photoreactive groups bound thereto. The photoreactive group(s) are preferably covalently bound, directly or indirectly, at one or more points along the nucleic acid. Such groups can be activated in order to attach the nucleic acid to the surface of a solid support, such as the surface of a chip. A photoreactive group of this invention is separate and distinct from whatever group or bonds within the nucleic acid may be susceptible to radiation. In turn, the photogroup provides a derivatized nucleic acid that can be selectively and specifically activated in order to attach the nucleic acid to a support, and in a manner that substantially retains its desired chemical or biological function.
As used herein, unless otherwise indicated, a xe2x80x9cphotoreactive compoundxe2x80x9d is a compound that is, or contains, one or more xe2x80x9cphotoreactive groupsxe2x80x9d, and can be used to derivatize a nucleic acid in order to form a xe2x80x9cphotoactivatable nucleic acid derivativexe2x80x9d having one or more photoreactive groups, directly or indirectly, covalently bound and pendent thereto. By xe2x80x9cdirectxe2x80x9d, its inflections, it is meant that the photoreactive compound is attached directly to the nucleic acid, whereas the xe2x80x9cindirectxe2x80x9d, and its inflections, will refer to the attachment of a photoreactive compound and nucleic acid to a common structure, such as a synthetic or natural polymer.
Applicants have discovered that photoreactive groups can be used to form derivatized nuleic acids, which in turn can be activated in order to attach the nucleic acids to the surface of a support in a manner that does not detrimentally affect the use of the immobilized nucleic acid for its intended purpose.
The present invention further provides a method of preparing such a composition, for instance, by derivatizing a nucleic acid with one or more photoreactive groups. The resultant photo-derivatized nucleic acid (e.g., oligonucleotide) can be covalently immobilized by the application of suitable irradiation, and usually without the need for surface pretreatment, to a variety of polymeric substrate surfaces. Thus, in one embodiment, the present invention provides a method that includes both the thermochemical attachment of one or more photoreactive groups to a nucleic acid and the photochemical immobilization of that nucleic acid derivative upon a substrate surface. This invention is particularly valuable for the fabrication of arrays of immobilized nucleic acids, e.g., by the use of printing or photolithographic techniques.
A particular advantage of covalently bonding nucleic acids onto surfaces, in a preferred embodiment described herein, is that the regions between the spots of immobilized nucleic acids remain hydrophobic, thereby providing clear separation between spots. There are also clear advantages of stable covalent bonds over adsorption. Stable bonds are important in applications where stringent hybridizations are needed or when amplification techniques, such as polymerase chain reaction (PCR), are used that involve thermocycling or where multiple probing is needed.
In another aspect, the invention provides a method of fabricating a nucleic acid probe array by the use of a photoactivatable nucleic acid composition as described herein. In yet another aspect, the invention provides a nucleic acid probe array fabricated by the use of one or more photoactivatable nucleic acid derivatives.
The photoactivatable nucleic acid derivative can take any suitable form, e.g., in the form of a single nucleic acid having one or more photogroups. In use, the photoactivatable nucleic acid derivatives of this invention provide a unique and convenient method for fabricating a nucleic acid probe array or for other methods involving nucleic acid immobilization.
Photoactivatable nucleic acid derivatives of the present invention can be provided in the form of one or more photoreactive groups bound (e.g., covalently attached directly or indirectly) to a nucleic acid. As used herein the term xe2x80x9cnucleic acidxe2x80x9d will include any of the group of polynucleotide compounds having bases derived from purine and from pyrimidine. Nucleic acids of particular use in the present invention include generally short, synthetic sequences more commonly known as oligonucleotides. The nucleic acid can be in any suitable form, e.g., single stranded, double stranded, or as a nucleoprotein. Examples of suitable nucleic acids include synthesized and/or natural molecules of deoxyribonucleic acid (DNA) (such as complementary DNA (cDNA)), ribonucleic acid (RNA), and peptide nucleic acid (PNA). PNA is a DNA mimic in which the native sugar phosphate DNA backbone has been replaced by a polypeptide. This substitution is said to increase the stability of the molecule, as well as improve both affinity and specificity.
One or more photogroups can be bound (e.g., directly) to the nucleic acid in any suitable fashion, e.g., by synthesizing an oligonucleotide, or derivatizing a natural or previously synthesized oligonucleotide, in such a manner as to provide a photogroup at the 3xe2x80x2-terminus, at the 5xe2x80x2-terminus, along the length of the oligonucleotide itself (e.g., pendent to an intermediate nucleotide or spacer within the nucleic acid), or any combination thereof.
The oligonucleotide component of a photoactivatable oligonucleotide composition can be synthesized using any suitable approach, including methods based on the phosphodiester chemistry and more recently, on solid-phase phosphoramidite techniques. See, generally, T. Brown and D. Brown, xe2x80x9cModem Machine-Aided Methods of Oligonucleotide Synthesisxe2x80x9d, Chapter 1, pp. 1-24 in Oligonucleotides and Analogues, A Practical Approach, F. Eckstein, ed., IRL Press (1991), the disclosure of which is incorporated herein by reference.
The stepwise synthesis of oligonucleotides generally involves the formation of successive diester bonds between 5xe2x80x2-hydroxyl groups of bound nucleotide derivatives and the 3xe2x80x2-hydroxyl groups of a succession of free nucleotide derivatives. The synthetic process typically begins with the attachment of a nucleotide derivative at its 3xe2x80x2-terminus by means of a linker arm to a solid support, such as silica gel or beads of borosilicate glass packed in a column. The ability to activate one group on the free nucleotide derivative requires that other potentially active groups elsewhere in the reaction mixture be xe2x80x9cprotectedxe2x80x9d by reversible chemical modifications. The reactive nucleotide derivative is a free monomer in which the 3xe2x80x2-phosphate group has been substituted, e.g., by dialkylphosphoramidite, which upon activation reacts with the free 5xe2x80x2-hydroxyl group of the bound nucleotide to yield a phosphite triester. The phosphite triester is then oxidized to a stable phosphotriester before the next synthesis step.
The 3xe2x80x2-hydroxyl of the immobilized reactant is protected by virtue of its attachment to the support and the 5xe2x80x2-hydroxyl of the free monomer can be protected by a dimethoxytrityl (DMT) group in order to prevent self-polymerization. A 2-cyanoethyl group is usually used to protect the hydroxyl of the 3xe2x80x2-phosphate. Additionally, the reactive groups on the individual bases are also protected. A variety of chemical techniques have been developed for the protection of the nucleotide exocyclic amino groups. The use of N-acetyl protecting groups to prepare N-acetylated deoxynucleotides has found wide acceptance for such purposes.
After each reaction, excess reagents are washed off the columns, any unreacted 5xe2x80x2-hydroxyl groups are blocked or xe2x80x9ccappedxe2x80x9d using acetic anhydride, and the 5xe2x80x2-DMT group is removed using dichloroacetic acid to allow the extended bound oligomer to react with another activated monomer in the next round of synthesis.
Finally, the fully assembled oligonucleotide is cleaved from the solid support and deprotected, to be purified by HPLC or some other method. The useful reagents and conditions for cleavage depend on the nature of the linkage. With ester linkages, as are commonly provided by linkage via succinyl groups, cleavage can occur at the same time as deprotection of the bases using concentrated aqueous ammonium hydroxide.
A composition of the present invention can be prepared by modifying nucleic acids using techniques within the skill of those in the art, given the present teaching. A review of methods for modifying nucleic acids is contained in Bioconjugate Chem., 3(1):165-186 (1990), the disclosure of which is incorporated herein by reference. Such methods include modifications introduced during chemical modifications of native or synthetic DNA, oligonucleotide synthesis, and enzymatic modifications.
In one embodiment of the present invention, nucleic acids, either natural or synthetic, can be derivatized with photoreactive groups randomly attached along the backbone or at either their 3xe2x80x2- or 5xe2x80x2- ends. For example, the bases present on the nucleotides making up the nucleic acid possess numerous reactive groups that can be derivatized using a heterobifunctional photoreactive compound having both a photoreactive group and a thermochemically reactive group suitable for covalent coupling to the bases. This approach will typically result in a relatively nonselective derivatization of the nucleic acid, both in terms of the location along the backbone as well as the number of photogroups per molecule.
In an alternative and more selective embodiment, a method of this invention can include the post-synthetic photoderivatization of an oligonucleootide which has had chemically reactive groups incorporated at specific sites during the synthesis, e.g. along the backbone or at either its 3xe2x80x2- or 5xe2x80x2- ends. For example, commercially available reagents or solid supports are available which permit the incorporation of amine groups at any of these locations in the oligonucleotide. These amine groups are then combined with a photoreactive compound having a photogroup and an N-oxysuccinimide ester (NOS), resulting in formation of an amide bond between the photogroup and the oligonucleotide. Those skilled in the art, given the present description, will appreciate the manner in which a variety of other reactions between electrophilic and nucleophilic species can provide similar coupling techniques. For example, the reaction between carboxylic acid chlorides with amines, or maleimide groups with sulfhydryl groups can be used to provide photoactivatible nucleic acid derivatives as well.
In another embodiment, one or more of the nucleotide building blocks typically used in oligo synthesis can themselves be derivatized with a reagent containing a photoreactive group by attachment of the reagent to one of the reactive functionalities present on the base residue of the nucleotide. The resulting derivatized nucleotide reagent can be used in an automated synthesizer, under conventional reaction conditions, in order to incorporate the photogroup at designated points along the chain or at either end of the oligonucleotide. In addition, commercially available non-nucleotide reagents, used for incorporation of chemically reactive groups as described above, can be reacted with the photoreactive compound to incorporate the photoreactive group, after which they can be used in the automated synthesizer to prepare the photoactivatible nucleic acids.
A variety of reagents are available for use in modifying nucleic acids, including those available under the trade name xe2x80x9cBiotin-Chem-Linkxe2x80x9d from Boehringer Mannheim (Indianapolis, Ind.). This cis-platinum reagent will bind to the N7 position of guanosine and adenosine bases. In a similar fashion, a photoreactive compound containing a cis-platinum moiety can be synthesized for use in photoderivatizing nucleic acids.
In another aspect, photoderivatized nucleotides can be synthesized and incorporated into nucleic acids using enzymatic techniques. For instance, a variety of reagents are available that can be used to label nucleic acids with biotin, fluorescein and digoxigenin (DIG). A nucleic acid can be labeled with a photoactivatable dideoxyribonucleotide or deoxyribonucleotide, using a terminal transferase, in order to provide either single or multiple photogroups, respectively, at the 3xe2x80x2-end of the nucleic acid.
Boehringer-Mannheim also sells a DIG-labeling kit called xe2x80x9cDIG-High Primexe2x80x9d for random primed labeling of DNA with DIG-11-UTP. xe2x80x9cBiotin High Primexe2x80x9d and xe2x80x9cFluorescein-High-Primexe2x80x9d products are also available. In a similar fashion, DNA can be random-primed labeled with a photoactivatable deoxyribonucleotide using the Klenow enzyme, as will become apparent to those skilled in the art.
DNA Polymerase I enzyme is commonly used for nick translation of DNA. By including photoactivatable deoxyribonucleotides in the mixture of deoxynucleotide triphosphates (xe2x80x9cdNTPsxe2x80x9d) the resulting polymerized product will contain one or more photoreactive groups along its length. Also, during Polymerase Chain Reaction (PCR) a photoactivatable deoxyribonucleotide can be included in the mixture of dNTPs for the labeling of amplification products. It is also possible to incorporate a photoribonucleotide into RNA, e.g., by the use of an RNA polymerase (e.g., SP6 or T7) and standard transcription protocols.
In a further embodiment of the invention, oligos can be derivatized with photoreactive groups by forming or attaching the intact oligo as a ligand along the backbone of a polymer that provides one or more photoreactive groups along its length, e.g., as described in co-pending U.S. patent application Ser. No. 08/916,913 for xe2x80x9cLatent Reactive Polymers with Biologically Active Moietiesxe2x80x9d, filed Aug. 15, 1997, the disclosure of which is incorporated herein by reference. A number of approaches can be used for the preparation of such a polymeric photo-oligo reagent. In one embodiment, the oligo can be prepared in monomer form by covalent attachment of a polymerizable vinyl group such as acryloyl to the oligo, either at the ends or along the backbone. This can be accomplished by reaction of acryloyl chloride with an amine derivatized oligo. These oligo monomers can then be copolymerized with a photoderivatized monomer along with other comonomers such as acrylamide or vinylpyrrolidone. The resulting polymer provides photogroups and oligos randomly attached along the backbone of the polymer. Alternatively, the polymer can be prepared with the photoreactive group at one end of the polymer by use of a chain transfer reagent having one or more photoreactive groups as part of the structure.
In yet a further embodiment, a photoreactive polymer (e.g., a preformed synthetic or naturally occuring polymer that itself is provided with photogroups) can be derivatized with oligos in order to form a composition of the present invention. In this approach, a polymer can be prepared, or modified, in order to have chemically reactive groups located along the backbone of the polymer, each of which is capable of reacting with appropriately substituted oligos. For example, polymers possessing activated groups such as NOS esters can be reacted with oligos containing amine functionality resulting in covalent attachment of the oligo to the polymer backbone through an amide bond. The polymeric portion itself can be derivatized with photogroups, either before, during or after its bonding to the oligo. For instance, a photoderivatized polymer can be prepared by polymerizing monomers that include photoderivatized monomers, or one or more photogroup can be added to the formed polymer in a manner similar to that described above with respect to oligos. Alternatively, polymers can be prepared having terminal photoreactive groups by the use of a chain transfer reagent having a photogroup as part of its structure. The oligo would then be added to the reactive groups in a second step.
The polymer backbone can be either synthetic or naturally occurring, and is preferably a synthetic polymer selected from the group consisting of oligomers, homopolymers, and copolymers resulting from addition or condensation polymerization. Naturally occurring polymers, such as polysaccharides and polypeptides, can be used as well. Preferred backbones are biologically inert, in that they do not provide a biological function that is inconsistent with, or detrimental to, their use in the manner described. Such polymer backbones can include acrylics such as those polymerized from hydroxyethyl acrylate, hydroxyethyl methacrylate, glyceryl acrylate, glyceryl methacrylate, acrylic acid, methacrylic acid, acrylamide and methacrylamide; vinyls such as polyvinyl pyrrolidone and polyvinyl alcohol; nylons such as polycaprolactam, polylauryl lactam, polyhexamethylene adipamide and polyhexamethylene dodecanediamide; polyurethanes; polyethers such as polyethylene oxide, polypropylene oxide, and polybutylene oxide; and biodegradable polymers such as polylactic acid, polyglycolic acid, polydioxanone, polyanhydrides, and polyorthoesters.
Photoreactive Groups
A preferred composition of this invention includes one or more pendent latent reactive (preferably photoreactive) groups covalently attached, directly or indirectly, to a nucleic acid. Photoreactive groups are defined herein, and preferred groups are sufficiently stable to be stored under conditions in which they retain such properties. See, e.g., U.S. Pat. No. 5,002,582, the disclosure of which is incorporated herein by reference. Latent reactive groups can be chosen that are responsive to various portions of the electromagnetic spectrum, with those responsive to ultraviolet and visible portions of the spectrum (referred to herein as xe2x80x9cphotoreactivexe2x80x9d) being particularly preferred.
Photoreactive groups respond to specific applied external stimuli to undergo active specie generation with resultant covalent bonding to an adjacent chemical structure, e.g., as provided by the same or a different molecule. Photoreactive groups are those groups of atoms in a molecule that retain their covalent bonds unchanged under conditions of storage but that, upon activation by an external energy source, form covalent bonds with other molecules.
The photoreactive groups generate active species such as free radicals and particularly nitrenes, carbenes, and excited states of ketones upon absorption of electromagnetic energy. Photoreactive groups may be chosen to be responsive to various portions of the electromagnetic spectrum, and photoreactive groups that are responsive to e.g., ultraviolet and visible portions of the spectrum are preferred and may be referred to herein occasionally as xe2x80x9cphotochemical groupxe2x80x9d or xe2x80x9cphotogroupxe2x80x9d.
Photoreactive aryl ketones are preferred, such as acetophenone, benzophenone, anthraquinone, anthrone, and anthrone-like heterocycles (i.e., heterocyclic analogs of anthrone such as those having N, O, or S in the 10- position), or their substituted (e.g., ring substituted) derivatives. Examples of preferred aryl ketones include heterocyclic derivatives of anthrone, including acridone, xanthone, and thioxanthone, and their ring substituted derivatives. Particularly preferred are thioxanthone, and its derivatives, having excitation energies greater than about 360 nm.
The functional groups of such ketones are preferred since they are readily capable of undergoing the activation/inactivation/reactivation cycle described herein. Benzophenone is a particularly preferred photoreactive moiety, since it is capable of photochemical excitation with the initial formation of an excited singlet state that undergoes intersystem crossing to the triplet state. The excited triplet state can insert into carbon-hydrogen bonds by abstraction of a hydrogen atom (from a support surface, for example), thus creating a radical pair. Subsequent collapse of the radical pair leads to formation of a new carbon-carbon bond. If a reactive bond (e.g., carbon-hydrogen) is not available for bonding, the ultraviolet light-induced excitation of the benzophenone group is reversible and the molecule returns to ground state energy level upon removal of the energy source. Photoactivatible aryl ketones such as benzophenone and acetophenone are of particular importance inasmuch as these groups are subject to multiple reactivation in water and hence provide increased coating efficiency.
The azides constitute a preferred class of photoreactive groups and include arylazides (C6R5N3) such as phenyl azide and particularly 4-fluoro-3-nitrophenyl azide, acyl azides (xe2x80x94COxe2x80x94N3) such as benzoyl azide and p-methylbenzoyl azide, azido formates (xe2x80x94Oxe2x80x94COxe2x80x94N3) such as ethyl azidoformate, phenyl azidoformate, sulfonyl azides (xe2x80x94SO2xe2x80x94N3) such as benzenesulfonyl azide, and phosphoryl azides (RO)2PON3 such as diphenyl phosphoryl azide and diethyl phosphoryl azide. Diazo compounds constitute another class of photoreactive groups and include diazoalkanes (xe2x80x94CHN2) such as diazomethane and diphenyldiazomethane, diazoketones (xe2x80x94COxe2x80x94CHN2) such as diazoacetophenone and 1-trifluoromethyl-1-diazo-2-pentanone, diazoacetates (xe2x80x94Oxe2x80x94COxe2x80x94CHN2) such as t-butyl diazoacetate and phenyl diazoacetate, and beta-keto-alpha-diazoacetates (xe2x80x94COxe2x80x94CN2xe2x80x94COxe2x80x94Oxe2x80x94) such as t-butyl alpha diazoacetoacetate. Other photoreactive groups include the diazirines (xe2x80x94CHN2) such as 3-trifluoromethyl-3-phenyldiazirine, and ketenes (xe2x80x94CHxe2x95x90Cxe2x95x90O) such as ketene and diphenylketene.
Upon activation of the photoreactive groups, the reagent molecules are covalently bound to each other and/or to the material surface by covalent bonds through residues of the photoreactive groups. Exemplary photoreactive groups, and their residues upon activation, are shown as follows.
The photoactivatable nucleic acids of the invention can be applied to any surface having carbon-hydrogen bonds, with which the photoreactive groups can react to immobilize the nucleic acids to surfaces. Examples of appropriate substrates include, but are not limited to, polypropylene, polystyrene, poly(vinyl chloride), polycarbonate, poly(methyl methacrylate), parylene and any of the numerous organosilanes used to pretreat glass or other inorganic surfaces. The photoactivatable nucleic acids can be printed onto surfaces in arrays, then photoactivated by uniform illumination to immobilize them to the surface in specific patterns. They can also be sequentially applied uniformly to the surface, then photoactivated by illumination through a series of masks to immobilize specific sequences in specific regions. Thus, multiple sequential applications of specific photoderivatized nucleic acids with multiple illuminations through different masks and careful washing to remove uncoupled photo-nucleic acids after each photocoupling step can be used to prepare arrays of immobilized nucleic acids. The photoactivatable nucleic acids can also be uniformly immobilized onto surfaces by application and photoimmobilization.
The invention will be further described with reference to the following non-limiting Examples. It will be apparent to those skilled in the art that many changes can be made in the embodiments described without departing from the scope of the present invention. Thus the scope of the present invention should not be limited to the embodiments described in this application, but only by embodiments described by the language of the claims and the equivalents of those embodiments. Unless otherwise indicated, all percentages are by weight.